This document summarizes research on identifying genetic factors associated with traumatic brain injury (TBI) outcomes. It notes that the ApoE4 allele has been associated with worse outcomes, but replication has been limited. Larger collaborative studies are needed using quantitative measures as "endophenotypes" to better detect genetic effects. Lessons from Alzheimer's disease research show that genome-wide association studies with thousands of patients are needed to reliably identify genetic risk factors for complex diseases like TBI.

1. Endophenotypes of TBI: Insights into Pathophysiology and Implications for Therapy Ramon Diaz-Arrastia, MD, PhD Director of Clinical Research Center for Neuroscience and Regenerative Medicine Professor of Neurology Uniformed Services University for Health Sciences

2. Neuroprotective Drugs in Animal Models GluR antagonists NMDA antagonists MK-801 Ketamine Dextromethorphan CGS19755 CPP Cerostat Selfotel CP 101-606 AMPA/KA antagonists CNQX KYNA GYKI-52466 Antioxidants Alpha tocopherol Tirilizad SOD1 Dexanabinol Anti-inflammatory IL1-RA TNF binding protein Soluble CR-1 receptor Anti-MAC-1 Minocycline Immunophilins (CsA, FK-506) Solumedrol Neurotrophins NGF bFGF IGF-1 NNZ-2566 EPO Miscellaneous TRH-related peptides Hypothermia Mg2+ CDP-Choline Glyburide Progesterone

3. Endophenotypes Endophenotype 1 Endophenotype 2 Phenotype Endophenotype 3 Endophenotype n

4. Endophenotypes of Myocardial Infarction Hypertension Hyperlipidemia Myocardial Infarction Vascular inflammation Endophenotype n

5. Endophenotypes of TBI Intracranial hemorrhage Inflammation Phenotype Diffuse Axonal Injury Endophenotype n

6. Subtypes of TBI DAI EDH Contusion/Hematoma SDH SAH/IVH Diffuse Swelling Courtesy of Alisa Gean, UCSF

7. Subtypes of TBI Identified by MRI ADC CT DWI FLAIR

8. Outline of Presentation Principles of Allelic Association Existing Allelic Association Studies in TBI Lessons from Alzheimer’s Disease field Future Genetic Studies in TBI Need for International TBI Genetics Consortium

9. Types of Genetic Association Studies Linkage analysis Extremely effective for single-gene diseases Caused by mutations (polymorphisms) that are very rare (< 0.01) but highly penetrant (high phenotypic risk ratio, > 10) Technology available for > 15 years Not well suited for complex diseases Multiple genes interact to produce phenotype Caused by polymorphisms that are common (> 0.1) but with a low phenotypic risk ratio (1.5 – 4) Environmental factors play a strong role Particularly when environmental factors are rare and stochastic

10. Types of Genetic Association Studies Allelic Association Studies Based on polymorphic alleles traveling with disease across families Can be done in family, case-control, or population-based samples Well-suited for studies on complex human diseases Environmental factors can be identified and controlled

11. Types of Genetic Association Studies Allelic Association Studies Candidate Gene Approach Feasible for past 10 years Many potential confounders Multiple hypothesis testing and publication bias Population stratification Phenotypic heterogeneity Most published studies are underpowered Replication has been poor

12. Types of Genetic Association Studies Allelic Association Studies Genome Wide Allelic Association (GWAS) Feasible over the past 4 years Human Genome Project and HapMap Project Requires far larger samples of cases and controls (n = 2,000 – 20,000) Requires sophisticated understanding of phenotypic variability of disease expression Endophenome/ The “Human Phenome” Project

15. Candidate Gene Studies in TBI Apolipoprotein E E4 allele associated with poor outcome in several studies Not uniformly reproducible Other gene variants associated with features of outcome DRD2, DAT, ACE, IL1RN, IL1A*2, ACE, COMT, p53, MAO-A, bcl-2, BDNF, Neprilysin, NGB1, 5HTT Very few have been confirmed None with genome-wide level of significance

16. Allelic Association of APOE4 in TBI

17. Negative Allelic Association of APOE4 in TBI

18. Weight of evidence supports an association between APOE4 and poor outcome after TBI. The association is modest and does not extend to all endophenotypes Mechanism of association is not known Allelic Association of APOE4 in TBI

19. Allelic Association of Candidate Genes in TBI

20. Multiple genes associated with poor outcome after TBI None of published studies reach genome-wide level of significance Besides APOE4, replication has been very sparse Allelic Association in TBI

21. Lessons from AD Research State of AD Genetics Research in 2007 Over 300genes had been associated in candidate gene studies with AD risk Most studies small. None reached genome-wide level of significance Only APOE was consistently replicated Since 2008, multiple GWAS studies have found reproducible associations with 10 additional genes

23. Guide selection of SNPs efficiently to “tag” common variants

24. Public release of all data (assays, genotypes)Phase I: 1.3 M markers in 269 people Phase II: +2.8 M markers in 270 people

26. BIN1

27. CLU

28. ABCA7

29. CR1

30. PICALM

31. CD33

32. MS4A4E

33. MS4A6E

35. Lessons from AD Genetics Research Number of TBI patients studied must increase by 1 order of magnitude 2,000 – 20,000 is standard these days Large international collaborative efforts are required Careful attention must be paid to quantitative endophenotypes

36. High Throughput Biomarker Assays Luminex-based Multiplex Immunoassays Rules Based Medicine, Inc. Austin, Texas O’Bryant et al, Arch Neurol 2010;67:1077-1081

37. Human MAP v.1.6--Antigens

38. Significant Analysis of Microarray (SAM) O’Bryant et al, Arch Neurol 2010;67:1077-1081

39. SAM 0 0 0 25 Wilcoxon test 1 Logistic regression 1 0 GRO alpha VDBP Venn Diagram of Analytical Methods O’Bryant et al, Arch Neurol 2010;67:1077-1081

41. GWAS with Quantitative Endophenotypes

42. Conclusions Genetic factors play a role in response of neural tissue to traumatic insults Good evidence for role of APOE Likely other genes also involved Current studies in TBI are underpowered by an order of magnitude Collaborative effort will be needed Attention to endophenotypes (imaging, biochemical, physiologic, psychometric) essential Please Email: Ramon.Diaz-Arrastia@usuhs.mil

43. Collaborators UT Southwestern Chris Madden Anne Hudak Mike Devous Roderick McColl Tony Whittemore Baylor Institute for Rehabilitation Stuart Yablon Mary Carlile Randi Dubiel Cindy Dunklin Libranda Callender Eva Wooster Tiffany Wren NIH R01 HD48179 U01 HD42652 R01 AG17861 US Dept. of Education H133A070027 UT Southwestern Caryn Harper Carol Moore Kan Ding Matthew Warner Catherine Oldenkamp Teddy Youn Lifang Peng Nasreen Sayed Christopher Paliotta Yale Gong UT Dallas Carlos Marquez de la Plata Jun-Yi Wang Khamid Bakhadirov Ana Arenivas Carlee Culver University of North Carolina Kirk Wilhelmsen Scott Chasse